Hydride transfer catalysed by Escherichia coli and Bacillus subtilis dihydrofolate reductase: coupled motions and distal mutations.

This paper reviews the results from hybrid quantum/classical molecular dynamics simulations of the hydride transfer reaction catalysed by wild-type (WT) and mutant Escherichia coli and WT Bacillus subtilis dihydrofolate reductase (DHFR). Nuclear quantum effects such as zero point energy and hydrogen tunnelling are significant in these reactions and substantially decrease the free energy barrier. The donor-acceptor distance decreases to ca 2.7 A at transition-state configurations to enable the hydride transfer. A network of coupled motions representing conformational changes along the collective reaction coordinate facilitates the hydride transfer reaction by decreasing the donor-acceptor distance and providing a favourable geometric and electrostatic environment. Recent single-molecule experiments confirm that at least some of these thermally averaged equilibrium conformational changes occur on the millisecond time-scale of the hydride transfer. Distal mutations can lead to non-local structural changes and significantly impact the probability of sampling configurations conducive to the hydride transfer, thereby altering the free-energy barrier and the rate of hydride transfer. E. coli and B. subtilis DHFR enzymes, which have similar tertiary structures and hydride transfer rates with 44% sequence identity, exhibit both similarities and differences in the equilibrium motions and conformational changes correlated to hydride transfer, suggesting a balance of conservation and flexibility across species.

Comparison of coupled motions in Escherichia coli and Bacillus subtilis dihydrofolate reductase.

Hybrid quantum/classical molecular dynamics simulations are used to compare the role of protein motion in the hydride transfer reaction catalyzed by Escherichia coli and Bacillus subtilis dihydrofolate reductase (DHFR). These two enzymes have 44% sequence identity, and the experimentally determined structures and hydride transfer rates are similar. The simulations indicate that the tertiary structures of both enzymes evolve in a similar manner during the hydride transfer reaction. In both enzymes, the donor-acceptor distance decreases to approximately 2.7 Angstroms at the transition state configurations to enable hydride transfer. Zero point energy and hydrogen tunneling effects are found to be significant for both enzymes. Covariance and rank correlation analyses of motions throughout the protein and ligands illustrate that E. coli and B. subtilis DHFR exhibit both similarities and differences in the equilibrium fluctuations and the conformational changes along the collective reaction coordinate for hydride transfer. A common set of residues that play a significant role in the network of coupled motions leading to configurations conducive to hydride transfer for both E. coli and B. subtilis DHFR was identified. These results suggest a balance between conservation and flexibility in the thermal motions and conformational changes during hydride transfer. Homologous protein structures, in conjunction with conformational sampling, enable enzymes with different sequences to catalyze the same hydride transfer reaction with similar efficiency.

Freezing a single distal motion in dihydrofolate reductase.

Constraining a single motion between distal residues separated by approximately 28 A in hybrid quantum/classical molecular dynamics simulations is found to increase the free energy barrier for hydride transfer in dihydrofolate reductase by approximately 3 kcal/mol. Our analysis indicates that a single distal constraint alters equilibrium motions throughout the enzyme on a wide range of time scales. This alteration of the conformational sampling of the entire system is sufficient to significantly increase the free energy barrier and decrease the rate of hydride transfer. Despite the changes in conformational sampling introduced by the constraint, the system assumes a similar transition state conformation with a donor-acceptor distance of approximately 2.72 A to enable the hydride transfer reaction. The modified thermal sampling leads to a substantial increase in the average donor-acceptor distance for the reactant state, however, thereby decreasing the probability of sampling the transition state conformations with the shorter distances required for hydride transfer. These simulations indicate that fast thermal fluctuations of the enzyme, substrate, and cofactor lead to conformational sampling of configurations that facilitate hydride transfer. The fast thermal motions are in equilibrium as the reaction progresses along the collective reaction coordinate, and the overall average equilibrium conformational changes occur on the slower time scale measured experimentally. Recent single molecule experiments suggest that at least some of these thermally averaged equilibrium conformational changes occur on the millisecond time scale of the hydride transfer reaction. Thus, introducing a constraint that modifies the conformational sampling of an enzyme could significantly impact its catalytic activity.

Effect of mutation on enzyme motion in dihydrofolate reductase.

Hybrid quantum-classical molecular dynamics simulations of a mutant Escherichia coli dihydrofolate reductase enzyme are presented. Although residue 121 is on the exterior of the enzyme, experimental studies have shown that the mutation of Gly-121 to valine reduces the rate of hydride transfer by a factor of 163. The simulations indicate that the decrease in the hydride transfer rate for the G121V mutant is due to an increase in the free energy barrier. The calculated free energy barrier is higher for the mutant than for the wild-type enzyme by an amount that is consistent with the experimentally observed rate reduction. The calculated transmission coefficients are comparable for the wild-type and mutant enzymes. The simulations suggest that this mutation may interrupt a network of coupled promoting motions proposed to play an important role in DHFR catalysis. This phenomenon has broad implications for protein engineering and drug design.